In the preparation of devices which can be used for processes such as electrophoresis, isoelectric focusing (IEF), and chromatography, it is often necessary to create liquid gradients. Such gradients can subsequently be immobilized (e.g., by a polymerization reaction which converts the liquid into a gel, or by using a liquid to coat a solid surface and then fully or partially drying the coated surface). Alternately, liquid gradients can be convection-stabilized (e.g., by adding the liquid gradient to a tube, trough, or other vessel which contains particulate matter or capillary tubes); such stabilized gradients will remain useful for a substantial period of time even though the compound forming the gradient remains liquid. As a third option, liquid gradients can be injected into or otherwise added to a container, conduit, or flow stream in a time-dependent manner, such as during various types of chromatography.
By controlling the starting compounds, liquid or gel gradients can involve various differing characteristics, such as varying concentration, varying density, varying acidity (usually expressed as pH), or varying molecular size. Such gradients are useful in many chemical and biological procedures, on scales ranging from analysis of sub-microgram quantities, to industrial production involving kilograms or metric tons.
As used herein, the term "liquid gradient" refers to a liquid output from a device, where the liquid output undergoes a transition from one state or condition (such as salt concentration, pH, density, molecular weight, etc.) to a different state or condition, where the transition is a predetermined, reproducible, and desirable function of time, volume, or some other variable. Liquid gradients can, in some cases, involve abrupt or steep transitions (which occur at "boundaries" or "interfaces" between different conditions or zones); they can also involve flat plateaus, or steep transitions. For example, a gradient with a steep or abrupt transition can be created by (1) partially filling a tube with a polymerizable compound, (2) halting the flow of liquid to the tube while the concentration of one or more substances in the liquid is substantially increased or decreased, (3) adding more liquid to the tube, and (4) polymerizing the liquid in the tube. That will create two zones of gel with an abrupt or steep boundary between them. If desired, either zone can have its own gradient.
In the prior art, liquid gradients used for electrophoresis, IEF, chromatography, etc., have been produced by several different means, none of which are wholly satisfactory.
One such means involves the delivery of liquid components from two or more separate vessels into a mixing chamber, using two or more pumps. One pump is controlled to provide an increasing flow rate of Liquid A, while the other pump provides a decreasing flow rate of Liquid B, so the total flow rate from both pumps remains constant or nearly constant. The flow rates can be adjusted in any way desired, to create linear, convex, or concave gradients.
That system has several shortcomings. It is complex and expensive; at least two variable-speed, accurately calibrated pumps and a control system are required, and the system must be run by a computer such as an Apple or an IBM-PC (which ties up the computer while the gradient is being formed). One such system, sold by Bodman Chemicals (Aston, PA) cost roughly two thousand dollars in March 1989, not including the computer. In addition, the use of pumps creates several potential problems. Pumps can become clogged (which is often difficult to detect promptly), they can cause fluctuations in flow rates which are also difficult to detect and correct, and most types of pumps except peristaltic pumps usually must be cleaned between uses, especially if different liquids are involved.
Another method of producing liquid gradients involves a mixing vessel which initially contains Liquid A, and a connected reservoir which contains Liquid B. The mixing vessel usually contains a stirring device. As liquid is removed from the mixing vessel and pumped to the device that will contain or process the gradient, it initially contains Liquid A only. As Liquid B is transferred from the reservoir to the mixing vessel, the resulting mixture will contain increasing concentrations of Liquid B.
These systems are sold in various configurations. For example, devices containing concentric cylinders (with a stirring device in the interior cylinder) are sold by Kontes Life Sciences Products (Vineland, NJ), Isolab Inc. (Akron, OH), Bethesda Research Labs (Gaithersburg, MD), and Whatman Lab Sales (Hillsboro, OR). "Dual piston" systems containing side-by-side cylinders (one of which contains a stirring device) are sold by Jule, Inc. (New Haven, CT), Cole-Parmer Instrument Co. (Chicago, IL), and Whatman Lab Sales.
There are various shortcomings to these dual reservoir systems. A stirring device normally must be used at high speed to ensure prompt and thorough mixing in the mixing chamber; however, this can cause splashing and fluid loss, and the vortex and turbulence can create abnormalities in the gradient. Two pumps are often required to run the system, and fluctuations or differences in the flow rates from those pumps can introduce variability in the gradient. Differences in the viscosity of the liquids can also introduce variability in the gradient. Those factors can make it very difficult to reproduce precisely equivalent gradients on different days, which is a serious drawback, since high levels of precision and reproducibility are often required for electrophoresis, IEF, or chromatography.
A third type of gradient maker is sold as the ID-300 Anderson-Dalt gradient maker by Hoefer Scientific Instruments (San Francisco, CA). This type of gradient maker, which is normally sold as part of a larger "Iso-Dalt" system used for two dimensional electrophoresis, is briefly described in N. G. Anderson and N. L. Anderson, Analytical Biochemistry 85: 341-543 (1978) at pages 344-345. Additional information is available in the 1988 ISO-DALT user's manual, available from Hoefer or from Large Scale Biology Corporation (Rockville, MD).
The Anderson-Dalt gradient maker involves a rectangular chamber made of clear acrylic. An internal barrier called a "septum," made of silicone rubber, divides the chamber into two compartments. By positioning the septum in an angled or curved configuration, it is possible to vary the sizes of the two compartments. For example, by placing the septum at an angle, Compartment A can have a large area and volume near the top of the rectangular chamber while Compartment B has a small area and volume; the relative sizes of the two compartments are reversed near the bottom of the chamber. Both compartments are drained, through tubes connected to an outlet at the bottom of each compartment, into a mixing device.
If compartment A is larger than compartment B at the top of the chamber, then a larger volume of liquid A than liquid B will enter the mixing device as the drainage process begins. This follows from two facts: (1) the two compartments establish and remain in hydrostatic equilibrium with each other (via the tubing) during the drainage process; therefore, (2) their fluid levels will remain roughly equal (dropping at the same rate) as both compartments are drained. At any moment, the ratio of the A:B mixture entering the mixing device will be equal to the ratio of the horizontal areas of their fluid surfaces. That ratio changes as the fluid level drops, due to the angle or curvature of the septum which divides the compartments. Since the positioning of the septum can be controlled, that ratio can be controlled, and the resulting mixture of A and B forms a controllable gradient.
That system also suffers from several disadvantages. It is expensive; in March 1989 the price of the gradient maker (excluding the rest of the Iso-Dalt system) was over a thousand dollars. It must be disassembled and cleaned before it can be used with different liquids. In some situations it may need to be autoclaved to ensure sterility, which can require substantial delays and can take up space, time, and energy in the autoclave unit. Furthermore, it can be difficult to reposition the septum, requiring various calculations to determine where the septum should be re-positioned and measurements to ensure that the resulting gradient is the one desired. That difficulty becomes even greater when curved rather than linear arrangements are desired. The chamber and septum are expensive to replace if broken or damaged, the pieces must be handled carefully, and the screws can be misplaced on a cluttered lab bench. The unit is relatively tall, and it is normally placed on an elevated base to generate enough hydrostatic pressure to deliver the liquids to the mixing device; both factors make it susceptible to being knocked over in a busy lab.
The object of this invention is to provide an improved system for creating liquid gradients. This system should be relatively inexpensive, and it minimizes or eliminates the need for pumps and computerized control units. It is also easy to assemble and use, resistant to breakage, and easily and quickly cleaned and converted between uses. Despite those conveniences, it is able to generate liquid gradients with a wide variety of profiles, in a precise and reproducible manner, with a minimum of calibration, calculation, or monitoring.